This invention pertains generally to the field of matrix-assisted laser desorption/ionisation (MALDI) and MALDI mass spectrometry (MS). More specifically, the present invention pertains to modified targets suitable for use with liquid matrices (e.g., glycerol and lactic acid) in liquid MALDI methods, as used, for example, in infrared (IR) liquid MALDI MS, preferably using time of flight (TOF) instruments. The present invention also pertains to ion sources, mass spectrometers, methods of MALDI and methods of mass spectrometry using such modified ion source targets.
Traditional mass spectrometric methods are extremely useful for the analysis of low molecular weight compounds. However, for high molecular weight compounds, for example, biopolymers such as proteins and carbohydrates, the problem to be solved was to convert relatively non-volatile macromolecules into intact, isolated, and ionised molecules in the gas phase. A number of so-called desorption/ionisation techniques have been developed to solve this problem. In field desorption methods, a strong electric field is applied to the sample. In fast atom bombardment and 252Cf plasma desorption, the sample is bombarded by highly energetic ions or atoms. In thermospray ionisation and electrospray ionisation methods, ions are generated directly from small, charged liquid droplets. Laser desorption/ionisation (LDI) and the newly developed variant of this method, xe2x80x9cmatrix assisted laser desorption/ionisationxe2x80x9d (MALDI), make use of short, intense pulses of laser light to induce the formation of intact gaseous ions.
Two factors dominate in the choice of laser for desorption methods. First, efficient and controllable energy transfer to the sample requires resonant absorption of the molecule at the laser wavelength. Consequently, lasers emitting in the ultraviolet (UV), which can couple to electronic states, or in the mid-infrared (mid-IR), which can excite rovibrational states, have so far shown the best results. Second, to avoid thermal decomposition, the energy must be transferred within a very short time. Typically, laser pulses or xe2x80x9cshotsxe2x80x9d with durations on the order of 1 to 200 ns are employed. Given the short pulse durations, and the fact that laser beams can easily be focussed to spot sizes that are small compared with the other dimensions of the ion source, the ions are generated essentially at a point source in space and time, as a xe2x80x9cpacketxe2x80x9d of ions. This pulsed desorption of ions favours the use of a time-of-flight (TOF) mass analyser, which makes it possible to record a complete mass spectrum for each laser shot. However, LDI methods may also be adapted for other mass spectrometers, including magnetic sector, quadrupole, Fourier transform ion cyclotron resonance (FT-ICR), and ion trap instruments.
In a time-of-flight (TOF) mass analyser, the velocity of an ion is used to determine its mass-to-charge ratio (m/z). A packet of ions is accelerated to a fixed kinetic energy by an electric potential, typically 1-30 kV. The velocity of a particular ion within the packet will then be proportional to (mi/zi)xe2x88x92xc2xd, where mi/zi is the ion""s mass-to-charge ratio. The ions are then allowed to pass through a field-free region, typically 0.1 to 3 m in length, where they are separated into a series of spatially discrete individual ion packets, each travelling with a velocity characteristic of its mass and charge. A detector at the end of the field-free region produces a signal as each ion packet strikes it. A recording of the detector signal as a function of time is a TOF mass spectrum. The difference between the start time, common to all ions, and the arrival time of an individual ion at the detector is proportional to (mi,zi)+xc2xd and therefore can be used to calculate the ion""s mass. Such a calculation can then be used to convert the axis of the spectrum from time into a mass-to-charge ratio axis, yielding a conventional mass spectrum.
The performance of mass spectrometers is typically described in terms of mass accuracy and mass resolving power. Mass accuracy is a measure of the error involved in assigning a mass to a given ion signal. It is typically expressed as the ratio of the mass assignment error divided by the mass o Of the ion and is frequently quoted as a percentage. Mass resolving power (also known as xe2x80x9cmass resolutionxe2x80x9d), m/xcex4m, is a measure of an instrument""s capability to produce separate signals from ions of similar mass. For TOF instruments, it is typically expressed as the mass, m, of a given ion signal divided by the full width of the signal, xcex4m, which is measured between the points of half-maximum intensity (FWHM). Factors which determine mass resolving power for a TOF instrument include the ion production time, initial velocity distribution, and extraction time. For example, conventional or xe2x80x9clinearxe2x80x9d TOF mass spectrometers may be adapted to include an xe2x80x9cion mirror,xe2x80x9d to yield a xe2x80x9creflectronxe2x80x9d TOF mass spectrometer (reTOF), which permits correction for the peak width contribution arising from the initial energy distribution. Reflectron configurations effectively increase the ion""s path length during separation, and therefore analysis time, and so increase susceptability to metastable effects.
The term xe2x80x9cmetastablexe2x80x9d is used herein in the conventional sense to describe ions which fragment at some time after formation and before detection, typically during mass analysis. Since most mass spectrometers rely on the separation of species according to mass and charge, the fragmentation of a large ion into two or more smaller species will change the separation parameters mid-flight. Consider, for example, a packet of ions, M1+, some of which decay in flight (in the field free region of a TOF instrument) to form (lighter) daughter ions, M2+, and neutral daughter species, M30. If, after fragmentation, the ions are subjected to an accelerating potential (e.g., an ion mirror in a reflectron instrument), then the parent and daughter ions will have different velocities. Both the parent ions, M1+, and the daughter ions, M2+, will be detected, but the latter at a mass intermediate between that of M1+ and M2+, according to the precise time of fragmentation and the accelerating potential. This can result in a smear or tail of intensity to lower mass (from M1+), and a consequent loss of resolving power. Metastable effects are largely dependent on the particular parent ion (e.g., greater metastable effects for labile and high mass ions), and the quantity and distribution of internal energy. Thus, ionization methods which deposit a large proportion of internal energy in levels which lead to bond-breaking and fragmentation often suffer from substantial metastable effects. For TOF instruments, metastable effects increase with increasing mass, since heavier ions have longer analysis times, and thus more opportunity to fragment before detection. For reflectron TOF (reTOF) instruments, the path length (and flight time) is also increased, again providing more opportunity for fragmentation prior to detection. Increasing background pressure, for example, in the field free region of a TOF instrument, have also been shown to increase metastable effects. See, for example, Berkenkamp, 1997.
Efforts to improve mass resolving power in TOF instruments have typically relied on focussing methods, to minimize the dependence of flight time on initial conditions. Examples of such methods include xe2x80x9cvelocity focussingxe2x80x9d (typically used when the spatial distribution is narrow) and xe2x80x9cspace focussingxe2x80x9d (typically used when the velocity distribution is narrow). See, for example, Vestal, 1998.
One method of xe2x80x9cvelocity focussingxe2x80x9d employs a delayed extraction of ions, as compared to a immediate and constant (static) extraction of ions. In xe2x80x9cstatic extractionxe2x80x9d methods, ions are subject to a large constant accelerating potential (e.g., 10-20 kV) from the instant they are formed. In xe2x80x9cdelayed extractionxe2x80x9d (DE) methods, also known as xe2x80x9ctime lag focussing,xe2x80x9d the application of the acceleration field is delayed for some time, xcex94t, after ion formation. For example, the ions may spend the first few hundred nanoseconds after formation in a field free environment, after which the acceleration potential is applied.
Another effort to improve resolving power for solid MALDI, by attempting to generate a more uniform velocity distribution through thermalisation of the ions, is described in U.S. Pat. No. 5,777,324 (Hillenkamp, 1998). This patent suggests the use of a cover, baffle, or compartment to impede or contain the plume formed during LDI within a baffle region, and so thermalise the ions before mass analysis. However, no data confirming the putative benefits (increased resolving power) for the proposed modifications are provided. A number of containment structures for solid MALDI are described. In one case, a box shaped compartment is constructed on top of the flat surface of the stage (rather than carved from the surface itself) with one or more exit apertures (FIGS. 3A and 3B, col. 5, line 61 through col. 6, line 11) with dimensions comparable to the that of the illumination spot, 10 to 500 xcexcm (col 5, line 61 through col. 6, line 11). However, no teaching is provided regarding the number, size, or arrangement of exit apertures. In another case, containment is effected using a non-conducting fibrous or porous sheath (FIGS. 9A and 9B, col. 8, line 48 through col. 10, line 16) formed from a pulp-based fibrous paper (such as laboratory filter paper), glass, ceramic, or polymeric materials. Other xe2x80x9copenxe2x80x9d containment structures include open tubes (FIG. 5A), a lean-to structure (FIG. 6A), open wells (FIGS. 11 and 12A), and pins (FIGS. 10 and 12B). In each case, the containment structure is within the ion extraction region, and is presumably subject to extensive field penetration/ distortion.
Laser desorption/ionisation (LDI) has been used in combination with mass spectrometry since the early 1960""s. In the mid-1980""s it was recognised that the upper limit on the mass of molecules that could be desorbed as intact ions (at that time, about 1000 for biopolymers) could be substantially increased by the use of a suitable matrix component, and xe2x80x9cmatrix-assisted LDIxe2x80x9d (MALDI) was born. See, for example, Hillenkamp et al., 1991a, 1991b, 1992.
Typically, as the technique now stands, a low concentration of analyte molecules, which usually exhibit only moderate absorption per molecule, is embedded in either a solid or liquid matrix consisting of small, highly absorbing species. Examples of solid matrices which have been shown to be effective include 2,5-dihydroxy benzoic acid (DHB), a mixture of DHB and 10% 5-methoxy salicylic acid, sinapinic acid, xcex1-cyano-4-hydroxycinnamic acid, nicotinic acid, 4-hydroxy picolinic acid, succinic acid, urea, and Tris buffer. Glycerol and lactic acid are examples of liquid matrices which have been used in IR MALDI. 3-Nitrobenzyl alcohol has been used as a liquid matrix for UV MALDI. Current reviews of MALDI methods are discussed in Bahr et al., 1994 and Hillenkamp et al., 1991c. Particularly with glycerol, users of MALDI have had to struggle with the problem of wanting enough matrix material to enhance LDI, but not so much that the matrix ion signal swamps out the analyte ion signal.
Although ultraviolet (UV) MALDI is now well established, infrared (IR) MALDI has enjoyed less popularity, primarily because of the high costs of infrared lasers and their limited availability in commercial MALDI instrumentation. However, recent publications have shown that IR MALDI is a valuable tool in the analysis of labile molecules such as phospho- and glycopeptides (Cramer, 1998) and RNA/DNA (Berkenkamp, 1998) and it can be anticipated that in the near future IR MALDI will be available as a standard option adding not more than 10-20% to the costs of a research grade MALDI TOF instrument.
IR MALDI offers a number of advantages over UV MALDI. In many cases, IR MALDI offers the advantage of being a xe2x80x9csofterxe2x80x9d method, as compared to UV MALDI, and is characterized by reduced metastable effects. This is despite the fact that apparently more of the absorbed laser energy goes into the analyte molecule in IR MALDI than in UV MALDI. One consequence of the softer nature of IR MALDI is that higher masses and labile biomolecules are accessible.
However, there are disadvantages inherent to the IR MALDI desorption process. The high penetration depth of infrared laser light in MALDI matrices promotes ablation rather than desorption leading, in the case of solid matrices, to a quick depletion of the irradiated sample spot and therefore the necessity, in many cases, to scan or raster across the whole sample. This clearly requires some skill, impairs mass accuracy due to space variation of the different desorption events, and is detrimental for automation.
One solution to this problem involves the use of liquid matrices. By using liquid matrices, an increased homogeneity of the analyte/matrix mixture can be achieved, the irradiated sample spot can replenish itself with sample, and therefore more successful desorption events can be obtained from the same spot at the same position in space. Several liquid matrices have been examined as possible matrices for IR MALDI, and in particular, glycerol. By using glycerol, and thus inducing only little internal energy, high mass and labile biomolecules can be detected intact, even in reflectron mode (which is more susceptible to metastable effects).
Successful detection of RNA and DNA containing more than a thousand nucleotides has been shown with glycerol (Berkenkamp, 1998; Kirpekar, 1999). However, it appears that glycerol exhibits different desorption characteristics than solid matrices. It has been reported (Feldhaus, 1999) that glycerol MALDI samples exhibit a higher threshold desorption energy at 2.94 xcexcm than succinic acid samples, although the absorption coefficient of glycerol is about a factor of ten higher. Analyte ion energy measurements showed a high initial energy dependence on the extraction field and a much broader initial energy distribution compared to IR MALDI solid matrices, such as succinic acid (Berkenkamp, 1999) and UV MALDI solid matrices, such as DHB. Increasing the extraction field, for example, increases the initial analyte ion energy as well. This might explain the poor IR MALDI results with glycerol using extraction fields of more than 1000-1500 V/mm (Cramer, 1997; Talrose, 1999), typically found in commercial instrumentation in reflectron mode. More successful results with glycerol have been demonstrated using extraction fields much lower than 1000 V/mm, often found in xe2x80x9chome-builtxe2x80x9d instruments. Further, the broader initial energy distribution observed with glycerol as matrix seems to restrict the maximum possible resolving power (Berkenkamp, 1999).
As discussed above, it has been shown that the xe2x80x9cstrengthxe2x80x9d or xe2x80x9chardnessxe2x80x9d of the extraction field has also been shown to influence the quality of spectra. Hard extraction fields (e.g., greater than about 1000 V/mm) are typical of commercial instrumentation, which employ a compact design with shorter extraction regions (e.g., xcx9c2 mm), while softer extraction fields (e.g., less than about 1000 V/mm, and typically less than about 500 V/mm) are often found in xe2x80x9chome-builtxe2x80x9d instruments which may employ longer extraction regions (e.g.,  greater than xcx9c5 mm) or lower ion extraction voltages (e.g.,  less than =10-15 kV)
In published glycerol-assisted IR LDI studies of peptides and proteins, laser desorption was initiated from thin layers (Berkenkamp, 1997, 1999; Talrose, 1999), frozen samples (Berkenkamp, 1997; Kraft, 1998), nitrocellulose substrates (Caldwell, 1998) or a combination of these. IR liquid MALDI from thick droplets has not yet been reported or it has been noted that spectra acquired from thick glycerol droplets are inferior (Talrose, 1999), primarily suffering from a lack of analyte ion signal and excessively high matrix ion signals. These observations can be interpreted as indications of significant interference of the bulk sample which is volatilised during the desorption process. Desorption from thin layers, frozen samples or porous surfaces could also restrict the total amount of evaporated sample which leads to successful detection of the analyte.
It has been known for years that the employment of liquid matrices (e.g., glycerol) in IR MALDI can be difficult, if not impossible, primarily due to the lack of analyte ion signal. Ion sources typically found in commercial MALDI instrumentation, which utilise high voltage, hard extraction fields ( greater than 1000 V/mm), are not well suited for IR liquid MALDI, and, to date, there have been no reports of IR liquid MALDI using instrumentation from one of the major MALDI TOF MS manufacturers.
The modified ion source targets of the present invention seek to ameliorate many of the recognised problems associated liquid MALDI MS, and in particular, IR liquid MALDI MS.
In conventional liquid MALDI methods, a drop of liquid sample is deposited on the flat surface of an unmodified target plate. In the present invention, liquid sample is deposited in a sample cavity formed in the outward facing target surface; the loaded cavity is then covered with a perforated sample cavity shield having one or more exit holes through which ions formed inside the sample cavity may escape or be extracted. Without wishing to be bound to any particular theory, it is postulated that the shielded sample cavity of the present invention permits both access for the incident laser beam, and means for escape or extraction of ions generated inside the sample cavity, while reducing the rate of evaporation of the liquid sample. Also, it is postulated that the desorption and collisional dynamics, particularly inside the sample cavity (in the gap, see below), lead to increased analyte ion signal and reduced matrix ion signal, while maintaining good resolving power.
Thus, certain embodiments of the present invention enjoy benefits such as increased sensitivity in analyte ion detection and reduced matrix ion signal compared to the non-modified commercial targets which are used in a commercial hard extraction ion sources, mass resolving power comparable to the best achievable with conventional IR MALDI or UV MALDI, and decreased sample volatility leading to a fivefold increase in analysis time compared to using conventional target designs (up to 1 hour using volumes of only 250 nL). Furthermore, the design of the modified ion source targets appears to be universal and easy to implement on most, if not all, conventional target plates.
Accordingly, objects of the present invention (one or more of which are met by certain embodiments) include the provision of a modified ion source target which is suitable for use in liquid MALDI MS methods, particularly IR liquid MALDI MS methods, and which:
(a) yields improved analyte ion signal;
(b) yields reduced matrix ion signal;
(c) permits a mass resolving power comparable to or better than that observed for solid MALDI MS methods;
(d) permits increased sampling time;
(e) permits the use of a relatively low background pressure;
(f) is suitable for use with desirable liquid matrices, such as glycerol and lactic acid.
(g) meets one or more of the above objects while using relatively short extraction regions and relatively high extraction voltages, such as those found in common commercially available instruments; and,
(h) meets one or more of the above objects, and which is a simple and inexpensive modification of a conventional ion source target.